This invention relates to a method and apparatus for determining the density of particles suspended in a fluid. More specifically, this invention relates to a method and apparatus for determining the particle density of a first type of particle suspended in a fluid having at least two types of particles suspended therein where the density of one is easily ascertained or is of standard value. The invention is particularly appropriate for determining a number corresponding to the count of platelets per unit volume of whole blood. Such number is often referred to as the density or concentration of platelets. The density or concentration may also be expressed in terms of particles per unit volume or in terms of hematocrit, that is fractional volume.
The examination of blood is one of the most common and most frequently used methods of medical diagnosis. Normal standard count values of red blood cells, white blood cells, platelets, and other constituent particles of blood and statistical distributions of such count values among healthy individuals have been established. These count values are commonly expressed as count per unit volume or density. Deviations from these standard values are often indicative of the presence of infectious diseases or other medical problems.
The platelet count per unit volume provides an important tool of clinical diagnosis in the examination of blood. Substantial deviation in the count per unit volume of platelets in the blood from a normal value is often indicative of the presence of disease. For example, tuberculosis often causes an increase in the platelet count, while other acute infectious diseases sometimes result in a sub-normal count. A greatly reduced platelet count is often indicative of acute leukemia. Mild drops in the platelet count may also be indicative of adverse drug effects.
Platelets are smaller, and exhibit a lower count, than red blood cells. Typically, normal human blood contains about five million red blood cells per microliter of whole blood and only about two hundred and fifty thousand platelets per microliter. The red blood cell is usually a biconcave disk of about eight microns in diameter and the platelet is a round, oval, or rod shaped particle having a diameter of about two and a half microns.
Because of their smaller size and fewer number compared to red blood cells, platelets are difficult to count in the presence of red blood cells. However, it is desirable to count platelets in the presence of red blood cells because special processes to divide out the platelets or to destroy the red blood cells prior to making a count of platelets, add delay and cost to the process and also reduce the reliability of the process.
In a traditional indirect method of ascertaining the density of platelets, platelets and red blood cells deposited on a microscope slide are counted simultaneously in a selected area, until some minimum number of red blood cells has been counted. The number of platelets is then obtained from the ratio of platelets to red blood cells and the more easily measured red cell density ascertained from a count obtained from a known volume of the same sample of blood.
In one such process, one part of whole blood is usually diluted in 99 parts of a physiological saline solution, such as ISOTON, a product of the Coulter Electronics Company. The mixture is then transferred to a slide and after 15 to 45 minutes, the slide is examined under a microscope. Sometimes the counting is accomplished with the aid of a counting chamber having a plurality of blocks of small squares. The number of particles counted in say ten such blocks is then multiplied by some constant dependent on the dilution ratio and block size. The platelets are visually counted simultaneously with red blood cells until 1,000 red cells have been counted in a common area of the slide. The number of platelets counted is, say, 68. The red cell count per unit volume, that is, the red cell density, will already be known or will become known as a result of other commonly required tests of the blood. For purpose of illustration, we will assume that the red cell density is 4,480,000 per microliter. This represents the actual density measured in a sample to which the invention has been applied.
The platelet density P.sub.D equals the red blood cell density RBC.sub.D multiplied by the ratio of platelets counted to red blood cells counted. In this example: EQU P.sub.D = RBC.sub.D .times. (68/1000) = 4,480,000 .times. 0.068 = 304,640 platelets per microliter.
This method offers the advantage of making the counting process independent of the sample volume as long as the volume of the available sample is large enough to provide a substantial number of platelets (preferably at least 50) and therefore a fairly accurate indication of platelet density. This method and other methods for counting platelets are described by pages 157 to 161 of Todd-Sanford Clinical Diagnosis by Laboratory Methods, Fourteenth Edition, edited by Israel Davidsohn and John Bernard Henry and published by W. B. Saunders Company, 1969.
Such a traditional indirect method of counting platelets and red blood cells simultaneously was either very time consuming or relatively inaccurate due to the paucity of the particles counted. For example in the above illustration, an error in the platelet count of only one platelet would result in a 1.5% error in the platelet density figure. Furthermore, the traditional method of calculating platelet density on the basis of a simultaneous count of red blood cells and platelets, required algebraic manipulation subsequent to completion of the counting process.
Electronic cell counters have made it possible to count particles in the blood in much shorter times with far greater accuracy than was ever possible using traditional microscope techniques.
The general concept of electronically counting different types of particles having distinguishing physical characteristics and suspended in a liquid sample, a known volume of which is passed through a sensing transducer, is old in the art. See for example, Coulter U.S. Pat. No. 2,656,508.
However, so far as is known, electronic particle counting has not been applied heretofore to the traditional indirect method of simultaneously counting red blood cells and platelets to determine the platelet count density of an undetermined volume of diluted whole blood. Furthermore, so far as is known, prior to the invention herein disclosed, there has been no known way of avoiding the algebraic manipulation required subsequent to the simultaneous count of red blood cells and platelets of a blood sample to calculate the platelet count per unit volume.
Though he concentration or density of particles in a fluid mediumis commonly expressed in terms of count per unit volume, it is also sometimes expressed in other ways, such as percent volume. More particularly, in the analysis of blood, routine measurements are often made of both red blood cell count and the hematocrit of the sample, that is its percent of blood volume occupied by red blood cells.
Precise reference values of hematocrit are usually made by a well-known centrifuging process.
In a well-known electronic process of measuring hematocrit the pulses to be counted are first weighted in proportion to the volumes of the respective particles being counted. Then the resultant weighted pulses are added, that is, integrated, thereby producing a signal that is proportional to the percent volume of the fluid that is occupied by the particles.
While the invention may be applied in other ways, for simplicity it will be described herein specifically in terms of density or concentration as represented by count per unit volume. Accordingly, it will be understood that the broad concepts that underlie the invention may be applied to density or concentration measured and expressed in other ways than in count per unit volume.